JPH04152883A - Method for culturing tryptophan synthase producing bacteria - Google Patents
Method for culturing tryptophan synthase producing bacteriaInfo
- Publication number
- JPH04152883A JPH04152883A JP27654790A JP27654790A JPH04152883A JP H04152883 A JPH04152883 A JP H04152883A JP 27654790 A JP27654790 A JP 27654790A JP 27654790 A JP27654790 A JP 27654790A JP H04152883 A JPH04152883 A JP H04152883A
- Authority
- JP
- Japan
- Prior art keywords
- culturing
- tryptophan synthase
- culture
- pressure
- producing bacteria
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010075344 Tryptophan synthase Proteins 0.000 title claims abstract description 21
- 238000012258 culturing Methods 0.000 title claims abstract description 15
- 241000894006 Bacteria Species 0.000 title claims abstract description 13
- 238000000034 method Methods 0.000 title claims description 18
- 230000000694 effects Effects 0.000 abstract description 9
- 230000000813 microbial effect Effects 0.000 abstract description 3
- 244000005700 microbiome Species 0.000 abstract description 3
- 241001646716 Escherichia coli K-12 Species 0.000 abstract 1
- 241000588724 Escherichia coli Species 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 5
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 5
- 108020004511 Recombinant DNA Proteins 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 229960004799 tryptophan Drugs 0.000 description 3
- 238000009423 ventilation Methods 0.000 description 3
- PLVPPLCLBIEYEA-WAYWQWQTSA-N (z)-3-(1h-indol-3-yl)prop-2-enoic acid Chemical compound C1=CC=C2C(\C=C/C(=O)O)=CNC2=C1 PLVPPLCLBIEYEA-WAYWQWQTSA-N 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- PLVPPLCLBIEYEA-UHFFFAOYSA-N indoleacrylic acid Natural products C1=CC=C2C(C=CC(=O)O)=CNC2=C1 PLVPPLCLBIEYEA-UHFFFAOYSA-N 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 description 1
- 239000000920 calcium hydroxide Substances 0.000 description 1
- 229910001861 calcium hydroxide Inorganic materials 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- -1 for example Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 235000010333 potassium nitrate Nutrition 0.000 description 1
- 239000004323 potassium nitrate Substances 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
し産業上の利用分野］
本発明は、トリプトファンシンターゼ生産菌の培養方法
に関するものであり、さらに詳しくは、トリプトファン
シンターゼが高活性で含有されている菌体を、高濃度で
、しかも安定的に培養する方法に関するものである。Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a method for culturing tryptophan synthase-producing bacteria. More specifically, the present invention relates to a method for culturing tryptophan synthase-producing bacteria. Moreover, it relates to a method for stably culturing.
［従来の技術］
Ｌ−トリプトファンを工業的に生産する方法は、従来か
ら様々な方法が検討されている。その内の一つに、トリ
プトファンシンターゼ生産菌の菌体を基質に作用させて
、Ｌ−トリプトファンを合成する方法（酵素法）かある
、この酵素法では、トリプトファンシンターゼ生産菌が
生産する酵素量を増強させる目的で、本菌株に組み換え
ＤＮＡ技術を施すことか行われている。[Prior Art] Various methods have been studied for industrially producing L-tryptophan. One of them is a method (enzymatic method) in which L-tryptophan is synthesized by allowing the cells of a tryptophan synthase-producing bacterium to act on a substrate.In this enzymatic method, the amount of enzyme produced by a tryptophan synthase-producing bacterium is For the purpose of enhancing this strain, recombinant DNA technology is being applied to this strain.
トリプトファンシンターゼ生産菌の組み換えＤＮＡ技術
の宿主としては、大腸菌か広く用いられているが、近年
の組み換えＤＮＡ技術の進歩に伴い、大腸菌の工業的規
模での培養方法が望まれている。Escherichia coli is widely used as a host for tryptophan synthase-producing bacteria using recombinant DNA technology, but with recent advances in recombinant DNA technology, a method for culturing Escherichia coli on an industrial scale is desired.
例えば、組み換えＤＮＡ技術により膏種された大腸菌を
培養するときに、制御可能なプロモーターを利用するこ
ともその一つである。For example, one example is the use of a controllable promoter when culturing E. coli that has been seeded using recombinant DNA technology.
［発明が解決しようとする課題］
しかしながら、トリプトファンシンターゼ生産菌の工業
的利用を考えた場合、トリプトファンシンターゼが高活
性で含有されていることのみならず、本菌株の高濃度の
培養を安定的に行えることが要求される。単に菌株を高
濃度で培養することのみが目的であれば、培地中の溶存
酸素が成育の律速とならないように留意して培養すれば
よいことが知られているが、トリプトファンシンターゼ
が高活性で含有されている菌体を、高濃度で、しかも安
定的に培養する方法は未だ知られていない。[Problems to be solved by the invention] However, when considering the industrial use of tryptophan synthase-producing bacteria, it is necessary to not only contain highly active tryptophan synthase but also to stably cultivate this strain at high concentrations. You are required to be able to do it. It is known that if the purpose is simply to culture a bacterial strain at a high concentration, it is sufficient to culture while paying attention to ensure that dissolved oxygen in the medium does not become rate-limiting for growth. There is still no known method for stably culturing the bacteria contained therein at a high concentration.
本発明は、以上のような課題を解決することを目的とす
る。すなわち、トリプトファンシンターゼが高活性で含
有されている菌体を、高濃度で、しかも安定的に培養す
る方法を提供することを目的とするものである。The present invention aims to solve the above problems. That is, the object of the present invention is to provide a method for stably culturing bacteria containing highly active tryptophan synthase at a high concentration.
［課題を解決するための手段］
本発明者らは、鋭意検討した結果、上記のような課題を
解決できる培養方法を見いだした６すなわち、本発明は
、培養缶体圧力（気相圧力）を０ 、８ ｋｇ／ ｃｍ
２ゲージ圧以下に保持し、好気的に培養することを特徴
とする、トリプトファンシンターゼ生産菌の培養方法を
提供するものである。[Means for Solving the Problems] As a result of intensive studies, the present inventors have discovered a culture method that can solve the above problems. 0,8 kg/cm
The present invention provides a method for culturing tryptophan synthase-producing bacteria, which is characterized by culturing aerobically while maintaining the pressure at 2 gauge pressure or less.
以下に、本発明をさらに詳細に説明する。The present invention will be explained in more detail below.
本発明に使用することのできる微生物は、組み換えＤＮ
Ａ技術により育種されたトリプトファンシンターゼ生産
菌であれば、とくに限定されるものではない。例えば、
トリプトファンシンターゼに対応する遺伝子をプラスミ
ド中にもつ、エシェリヒア、コリ（Ｅｓｅｈｅｒｉｅｈ
ｉａ ｃｏｌｉ）Ｋ−１２ＹＫ２００９（ＦＥＲＮ Ｐ
−７９５７＞、エシェリヒア・コリに−１２ＹＫ２０１
４（ＦＥＲＮ Ｐ−８３２８）、エシェリヒア・コリに
−１２Ｙに２００４（ＦＥＲＮ ＢＰ−１７３２）など
が好適に用いられる。Microorganisms that can be used in the present invention include recombinant DNA
There is no particular limitation as long as it is a tryptophan synthase producing bacterium bred by technique A. for example,
Escherichia coli has a gene corresponding to tryptophan synthase in its plasmid.
ia coli) K-12YK2009 (FERN P
-7957>, Escherichia Cori -12YK201
4 (FERN P-8328), 2004 (FERN BP-1732) for -12Y for Escherichia coli, etc. are preferably used.
前記微生物の培養方法を以下に述べる。培地は、炭素源
として、グルコース、グリセロール、フラクトース、シ
ュクロース、糖蜜等の種々の炭水化物を使用することが
できる。これらの炭素源は、もちろん、成育の律速およ
び増殖阻害を起こさない範囲で逐次添加することが望ま
しい、また、窒素源としては、塩化アンモニウム、硫酸
アンモニウム、リン酸アンモニウム等のアンモニウム塩
、硝酸ナトリウム、硝酸カリウム、硝酸アンモニウム等
の硝酸塩、アンモニア等を例示することができる。無機
物としては、例えば、リン酸カリウム、ＷＲ酸マグネシ
ウム、鉄、マンガン、亜鉛等が挙げられる。さらに、必
要に応じて、ビタミン、アミノ酸等の栄養源を添加する
こともできる。The method for culturing the microorganism will be described below. The culture medium can use various carbohydrates such as glucose, glycerol, fructose, sucrose, molasses, etc. as carbon sources. Of course, it is desirable to add these carbon sources sequentially to the extent that they do not limit growth or inhibit growth.As nitrogen sources, ammonium salts such as ammonium chloride, ammonium sulfate, and ammonium phosphate, sodium nitrate, and potassium nitrate can be added. , nitrates such as ammonium nitrate, ammonia, and the like. Examples of the inorganic substances include potassium phosphate, magnesium WR acid, iron, manganese, and zinc. Furthermore, nutritional sources such as vitamins and amino acids can be added as necessary.
培養温度は、通常２０〜４５℃、好ましくは３０〜４０
℃の範囲内がよい、培養中の培地の９Ｈは、一般に６〜
９、好ましくは７〜８の範囲内が適しており、アルカリ
性物質を培地に添加することによって、ｌ１１−１を調
節することができる。The culture temperature is usually 20 to 45°C, preferably 30 to 40°C.
The 9H of the culture medium is generally within the range of 6 to
9, preferably within the range of 7 to 8, and l11-1 can be adjusted by adding an alkaline substance to the medium.
このアルカリ性物質としては、例えば、アンモニア、水
酸化ナトリウム、水酸化カリウム、水酸化カルシウム笠
の水溶液が好適に用いられる。As this alkaline substance, for example, aqueous solutions of ammonia, sodium hydroxide, potassium hydroxide, and calcium hydroxide are preferably used.
また、培養缶体圧力（気相圧力）の制御方法としては１
缶体の排気量および／または通気量を調節することによ
り行い、ゲージ圧として、０．８Ａｇ／ｃｗ”以下、好
ましくはＯ，，２〜０．５ ｋｇ／ ｃｍ２に保持しつ
つ培養を行うのがよい、このとき、培地中の溶存酸素濃
度（ＤＯ）が減少して、菌体増殖の律速となる場合には
、培養缶体圧力を０ 、８ ｋｇ／ ｃｍ”以下の範囲
で段階的に上昇させてもよく、さらに、通気量をｌｖｖ
ｍ以上にしたり、酸素ガスを通気すること、また培養槽
に応じて設定されている撹拌数を増加させること等によ
り、対応することが可能である。In addition, as a method of controlling the culture vessel pressure (gas phase pressure), 1
Cultivation is carried out by adjusting the exhaust volume and/or ventilation volume of the can, and the gauge pressure is maintained at 0.8 Ag/cw" or less, preferably 0.2 to 0.5 kg/cm2. At this time, if the dissolved oxygen concentration (DO) in the medium decreases and becomes the rate-limiting factor for cell growth, reduce the culture vessel pressure in stages within the range of 0.8 kg/cm" or less. In addition, the ventilation amount may be increased by lvv.
It is possible to deal with this by increasing the number of stirring points or more, by aerating oxygen gas, or by increasing the number of stirrings set depending on the culture tank.
他の培養条件としては、通常用いられている方法をその
まま応用することができる１例えばトリプトファンシン
ターゼをコードする遺伝子が、トリプトファンプロモー
ターにより発現調節されている場合には、効率よく発現
させるために、培養中にインドールアクリル酸を添加す
ることも可能である。As for other culture conditions, commonly used methods can be applied as they are.1 For example, if the gene encoding tryptophan synthase is expression-regulated by the tryptophan promoter, culture conditions are necessary to ensure efficient expression. It is also possible to add indole acrylic acid therein.
［実 施 例コ 以下に本発明を実施例により説明する。[Example of implementation] The present invention will be explained below using examples.
実」１倒」。Real "1 defeat".
（前培養）
下記第１表に示す組成の培地５Ｑｍｌ！を、５００ｍ１
容三角フラスコに分注し、１２０℃、１５分間滅菌処理
を行い、エシェリヒア・コリに一１２ＹＫ２００９（Ｆ
ＥＲＮ Ｐ〜７９５７）を−白金耳植菌した。さらに１
２０℃で１０分間滅菌した５０％（ｗｔ／ｖｏｌ）グル
コース溶液２ｍｌを添加した後、３７℃にて１日間、振
盪培養して、前項１物を得た。(Preculture) 5Qml of medium with the composition shown in Table 1 below! , 500m1
Dispense into Erlenmeyer flasks, sterilize at 120°C for 15 minutes, and incubate Escherichia coli with 112YK2009 (F
ERNP~7957) was inoculated into a platinum loop. 1 more
After adding 2 ml of 50% (wt/vol) glucose solution sterilized at 20°C for 10 minutes, the mixture was cultured with shaking at 37°C for 1 day to obtain Item 1 above.
（後培養）
前培養に用いた、下記第１表に示す組成の培地１５１を
１２０℃、１５分間滅菌処理し、これを３０１容の通気
撹拌槽に入れ、滅菌済５０％（―ｔ／ｖｏｌ）グルコー
ス溶液６００ｚ１を添加後、前培養物３００ｍ１を植菌
し、３４℃、ｐＨ７，２（２５％アンモニア水で調整）
で培養した。なお、グルコースは、培養液中の濃度が０
．１〜２（ｗｔ／ｖｏｌ）％となるように逐次添加した
。撹拌回転数は、５００ｒｐｍ、通気量は、１５ＮＮ／
分に調整した。培養開始６時間後に、インドールアクリ
ル酸を８０Ｈ／ｅの濃度になるように添加した後、下記
第２表に示すそれぞれの培養缶体圧力を維持しながら培
養を行った。 培養２０時間後に培養を終了し、培養液
’５００ｚｌから遠心分１（８０００ｒｐｍ、２０分間
、４℃）により回収した湿菌体のトリプトファンシンタ
ーゼ比活性を測定した。なお、トリプトファンシンター
ゼ比活性は、回収した湿菌体を１００１１Ｍトリス塩酸
Ｍ衝液（ｐＨ７，８）にて−度洗浄後、この湿菌体２０
０ｍｇを秤量し、さらに前記Ｍ新液１１１を添加した後
、菌体破壊装置［ソニファイヤー（ｓｏｎｉｆｉｅｒ）
２５０、ブランソン（ＢＲＡＮＳＯＮ）製］にて菌体を
破壊したのち、遠心分１！ｉ（１２０Ｏｒｐｍ、２０分
間、４℃）により得た上澄液を常法、すなわちヤノフス
キ（Ｙａｎｏｆｓｋｙ）らの方法（メソッドイン・エン
ザイモロジー（Ｍｅｔｈｏｄ ｉｎ Ｅｎｚｙｍｏｌｏ
ｇｙ）、νｏ１．５． ７９４頁、１９６２年参照）に
よりトリプトファンシンターゼ活性を測定して算出した
。(Post-culture) The medium 151 used for pre-culture and having the composition shown in Table 1 below was sterilized at 120°C for 15 minutes, placed in a 301-volume aeration stirring tank, and sterilized at 50% (-t/vol). ) After adding 600 z1 of glucose solution, inoculate 300 ml of preculture and maintain at 34°C, pH 7.2 (adjusted with 25% aqueous ammonia).
It was cultured in Note that glucose has a concentration of 0 in the culture solution.
．． It was added sequentially so that it became 1-2 (wt/vol)%. The stirring rotation speed is 500 rpm, and the ventilation rate is 15 NN/
Adjusted to minutes. Six hours after the start of culture, indole acrylic acid was added to a concentration of 80 H/e, and culture was carried out while maintaining the respective culture vessel pressures shown in Table 2 below. After 20 hours of culture, the culture was terminated, and the tryptophan synthase specific activity of the wet bacterial cells collected from the 500 zl culture solution by centrifugation 1 (8000 rpm, 20 minutes, 4°C) was measured. The tryptophan synthase specific activity was determined by washing the collected wet bacterial cells twice with a 10011M Tris-HCl solution (pH 7,8).
After weighing 0 mg and further adding the M new solution 111, a microbial cell destruction device [sonifier]
250, manufactured by BRANSON], and then centrifuged for 1 minute. The supernatant obtained by i (120 Orpm, 20 minutes, 4°C) was subjected to a conventional method, that is, the method of Yanofsky et al. (Method in Enzymolo
gy), νo1.5. It was calculated by measuring the tryptophan synthase activity (see p. 794, 1962).
その結果を第２表に示す。The results are shown in Table 2.
第 １ 表
ｐＨ
７，２
第
つ
表
活性は、各培養缶体圧力において５回繰り返し、培養し
た菌体のトリプトファンシンターゼ比活性の平均値を、
培養缶体圧力１ｋｇ／ｃｍ’のときの値を１００とした
相対値で表した。Table 1: pH 7,2 Table 2: Activity is the average value of the tryptophan synthase specific activity of the cells cultured five times at each culture vessel pressure.
It is expressed as a relative value with the value when the culture can pressure is 1 kg/cm' as 100.
人１ぢ副
菌株をエシェリヒア・コリに−１２ＹＫ−２０１４（Ｆ
ＥＲＮＰ−８３２８＞を使用する以外は、実施例１と同
様の実験を行った。結果を第３表に示す。-12YK-2014 (F
An experiment similar to Example 1 was conducted except that ERNP-8328> was used. The results are shown in Table 3.
第 表 活性は、実施例１と同様の定義で表した。No. table Activity was expressed using the same definition as in Example 1.
第２表および第３表から、培養缶体圧力が０．８ｋｉ／
ｃｍ２以下のとき、相対活性が高くなることが判る。From Tables 2 and 3, the culture vessel pressure is 0.8 ki/
It can be seen that the relative activity becomes high when it is below cm2.
［発明の効果］
本発明方法は、トリプトファンシンターゼが高活性で含
有されている菌体を高濃度で、しがち安定的に培養する
ことができる効果がある。[Effects of the Invention] The method of the present invention has the advantage that microbial cells containing highly active tryptophan synthase can be stably cultured at a high concentration.
Claims (1)
ジ圧以下に保持し、好気的に培養することを特徴とする
、トリプトファンシンターゼ生産菌の培養方法。A method for culturing tryptophan synthase-producing bacteria, which comprises maintaining the culture vessel pressure (gas phase pressure) below 0.8 kg/cm^2 gauge pressure and culturing aerobically.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP27654790A JPH04152883A (en) | 1990-10-17 | 1990-10-17 | Method for culturing tryptophan synthase producing bacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP27654790A JPH04152883A (en) | 1990-10-17 | 1990-10-17 | Method for culturing tryptophan synthase producing bacteria |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04152883A true JPH04152883A (en) | 1992-05-26 |
Family
ID=17571007
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP27654790A Pending JPH04152883A (en) | 1990-10-17 | 1990-10-17 | Method for culturing tryptophan synthase producing bacteria |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04152883A (en) |
-
1990
- 1990-10-17 JP JP27654790A patent/JPH04152883A/en active Pending
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